68 HEMOGLOBIN 



constantly present. If the carbon dioxide tension is permitted to fall, part of the 

 oxyhaemoglobin is redissolved as alkali salt. 



The purity of the oxyhaemoglobin obtained by the present method has been con- 

 trolled by a determination of the ratio of the oxyhaemoglobin present, as determined 

 by Van Slyke and Stadie's procedure (8), to the total haemoglobin pigments present, 

 determined as cyanhaemoglobin by Stadie's method (9). . .preparations of 96 to 100 

 per cent, of the theoretical oxygen capacity were obtained. The relative freedom of 

 the product from salts was controlled by conductivity measurements of saturated 

 aqueous solutions. 



(3) Parsons has very kindly given me a description of his method 

 in the following words : 



Starting with the observation that a frozen solution of haemoglobin consists of a 

 mixture of haemoglobin crystals and ice crystals, we conceived the idea that if a 

 solution of haemoglobin were centrifuged while it was being frozen the crystals of ice 

 and haemoglobin would be separated and the latter would collect at the bottom of 

 the containing tube. We soon proved that this separation does in fact take place 

 under these circumstances, but found the method very difficult to apply as even 

 when the centrifuge was installed in a refrigerating chamber it was very difficult to 

 cool it at a rate sufficient to counterbalance its own rate of production of heat by 

 friction. We therefore later adopted the simpler converse process of centrifuging a 

 frozen haemoglobin solution while it was allowed to thaw, and in this way obtained 

 a somewhat slower but easier method of concentrating and crystallising the pigment. 

 We find that Offringa (10) had previously observed the crystallisation of horse haemo- 

 globin during the centrifuging of its frozen solution, but we find further that this 

 process can be used for the crystallisation of the more soluble haemoglobins such 

 as those of ox and sheep (so that it possesses the great advantage that ordinary 

 slaughterhouse blood can be used for the preparation), and that it can also be used 

 for the crystallisation of the corresponding "reduced" CO- and methaemoglobins 

 of these animals, and for the concentration of haemocyanin and similar animal pig- 

 ments. The practical details of the method may be stated quite briefly. The corpuscles 

 are freed from serum proteins by repeated washings in the centrifuge with 1 per cent, 

 sodium chloride solution (which, being slightly hypertonic, causes shrinkage and 

 better settlement). The corpuscle mass, sucked as free as possible from salt solution, 

 is then laked by freezing and thawing repeated twice or three times if necessary. 

 The resulting liquid is placed in a bottle and the stromata are removed from it by 

 vigorous shaking with about 1/10 of its volume of well-washed asbestos pulp for 2 to 

 3 hours. According to a suggestion made by Bayliss the gradual removal of the 

 stromata on to the surface of the asbestos can be followed by observing the gradual 

 decrease of the viscosity of the solution. The asbestos is removed by centrifuging 

 and the solution of haemoglobin is divided among the centrifuge tubes which, after 

 balancing, are placed in the refrigerator or freezing mixture in order that their con- 

 tents may freeze. The freezing should be carried out at a temperature in the neigh- 

 bourhood of — 5° to — 7° C, i.e. well above — 22° C, which is the cryohydric point 

 at which crystals of solid sodium chloride separate out. When the freezing is com- 

 plete the tubes are placed in the machine and rotated at a speed of at least 4000 



